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Proapoptotic Function of the Retinoblastoma Tumor Suppressor Protein

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Proapoptotic Function of the Retinoblastoma Tumor Suppressor Protein View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cancer Cell Article Proapoptotic Function of the Retinoblastoma Tumor Suppressor Protein Alessandra Ianari,1,2 Tiziana Natale,2 Eliezer Calo,1 Elisabetta Ferretti,2 Edoardo Alesse,3 Isabella Screpanti,2 Kevin Haigis,4 Alberto Gulino,2,5,* and Jacqueline A. Lees1,* 1David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA 2Department of Experimental Medicine, La Sapienza University of Rome, 00161 Rome, Italy 3Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy 4Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA 5Neuromed Institute, 86077 Pozzilli, Italy *Correspondence: [email protected] (A.G.), [email protected] (J.A.L.) DOI 10.1016/j.ccr.2009.01.026 SUMMARY The retinoblastoma protein (pRB) tumor suppressor blocks cell proliferation by repressing the E2F transcrip- tion factors. This inhibition is relieved through mitogen-induced phosphorylation of pRB, triggering E2F release and activation of cell-cycle genes. E2F1 can also activate proapoptotic genes in response to geno- toxic or oncogenic stress. However, pRB’s role in this context has not been established. Here we show that DNA damage and E1A-induced oncogenic stress promote formation of a pRB-E2F1 complex even in prolif- erating cells. Moreover, pRB is bound to proapoptotic promoters that are transcriptionally active, and pRB is required for maximal apoptotic response in vitro and in vivo. Together, these data reveal a direct role for pRB in the induction of apoptosis in response to genotoxic or oncogenic stress. INTRODUCTION In normal cells, pRB’s repressive activity is controlled by its cell-cycle-dependent phosphorylation (Trimarchi and Lees, The retinoblastoma gene (RB1), a member of the pocket protein 2002). In response to mitogenic signaling, pRB is sequentially family with p107 and p130, was the first known tumor phosphorylated by the Cdk complexes cyclin D-Cdk4/6 and suppressor. The retinoblastoma protein (pRB) is targeted by cyclin E-Cdk2. This phosphorylation is sufficient to induce pRB the transforming proteins of the DNA tumor viruses (e.g., adeno- to release E2F, thereby allowing activation of E2F-responsive viral E1A), and it is functionally inactivated in a large proportion genes in late G1. However, phosphorylated pRB (ppRB) persists of human tumor cells due to mutations of either the RB1 gene in the nucleus through the remainder of the cell cycle until it is itself or its upstream regulators (Trimarchi and Lees, 2002). dephosphorylated by protein phosphatase 1 at the end of pRB’s tumor-suppressive activity is thought to be largely depen- mitosis (Ludlow et al., 1993). It is widely assumed that ppRB is dent upon its ability to directly bind members of the E2F family of functionally inactive and that dephosphorylation restores pRB transcription factors and prevent them from promoting transcrip- to the active state. The majority of human tumors carry mutations tion of genes required for cell proliferation (Trimarchi and Lees, that disable pRB-mediated repression of E2F (Sherr and McCor- 2002). This inhibition can occur via two distinct mechanisms: mick, 2002). These mutations either inactivate the RB1 gene pRB binds to sequences within E2F’s transactivation domain itself or promote pRB phosphorylation in the absence of normal and inhibits its function, and the resulting pRB-E2F complex mitogenic signals through activation of the cyclin D-Cdk4/6 recruits a number of transcriptional corepressors, including kinases or inactivation of the Cdk inhibitor p16. These changes histone deacetylases (HDACs), methyltransferases, and poly- result in the inappropriate release of E2F, thereby inducing tran- comb group proteins to actively repress the promoters of E2F scriptional activation of E2F target genes and consequently cell target genes. proliferation. SIGNIFICANCE Retinoblastoma protein (pRB) function is disrupted in many human tumors through either inactivation of the RB1 gene or alterations in its upstream regulators. pRB’s tumor-suppressive activity is at least partially dependent upon its ability to arrest cells through E2F inhibition. Our data here now establish a second role for pRB as a stress-induced activator of apoptosis. Notably, pRB’s ability to promote either arrest or apoptosis seems to be context dependent, with apoptosis being favored in proliferating cells. This finding has the potential to explain why cells are typically more resistant to apoptosis when in the arrested state. Most importantly, our observations suggest that RB1 status will influence tumor response to chemotherapy by impairing both the arrest and apoptotic checkpoint responses. 184 Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc. Cancer Cell pRB Induces Apoptotic Genes in Response to Stress It is well established that E2F1, among other E2F family that the observed DNA damage-induced formation of the pRB- members, also contributes to the induction of apoptosis in E2F1 complex is neither dependent on p53 nor simply an indirect response to either DNA damage or oncogenic stress (Iaquinta consequence of a G1 arrest. and Lees, 2007). This is thought to be a critical event in suppress- pRB’s ability to bind to E2F is normally limited to the early ing the formation of tumors. Work from many laboratories has stages of the cell cycle when pRB exists in the hypophosphory- shown that genotoxic stress induces E2F1 recruitment to the lated form. Therefore, it was surprising to observe pRB-E2F1 promoters of proapoptotic genes including p73 and Caspase complexes in a population highly enriched for G2/M phase cells. 7, coincident with their transcriptional activation, even in cells To more directly address the influence of cell-cycle phasing, we that retain wild-type pRB (Pediconi et al., 2003). This led us to used serum deprivation and readdition to generate two popula- consider how pRB influences DNA damage-induced apoptosis. tions of T98G cells that were greatly enriched for either G0/G1 The prevailing view is that pRB is an antiapoptotic regulator. (70%) or proliferating (95% S or G2/M phase) cells (Figure 1C) Early support for this model came from the finding that several and then treated these with doxorubicin. Consistent with the tissues in Rb mutant mice display both ectopic proliferation known cell-cycle-dependent phosphorylation of pRB, the pRB and apoptosis (Jacks et al., 1992). However, it is now clear protein was present in its slower mobility form in untreated that much of this apoptosis is non-cell autonomous, resulting proliferating cells, and it bound little E2F1 (Figure 1C). Notably, from a proliferation defect in the extraembryonic tissues (de doxorubicin treatment was still able to induce formation of the Bruin et al., 2003; Wenzel et al., 2007; Wu et al., 2003). Analysis pRB-E2F1 complex in this proliferating population (Figure 1C). of tissue-specific Rb mutant models reinforces the notion that This occurred independently of any change in total levels of pRB plays a much more nuanced role in apoptosis. Loss of E2F1 protein (Figure 1C), and it correlated with full activation of pRB in neuronal tissue (MacPherson et al., 2003), lung (Mason- the apoptotic program as judged by PARP-p85 induction (data Richie et al., 2008; Wikenheiser-Brokamp, 2004), skin (Ruiz not shown). Importantly, the G0/G1 cells within the proliferating et al., 2004), and intestine (Haigis et al., 2006; Wang et al., population cannot fully account for this DNA damage-induced 2007) drives ectopic proliferation but has no effect on apoptosis. pRB-E2F1 complex because the treated proliferating cells and In contrast, Rb inactivation in the lens (de Bruin et al., 2003) and untreated G0/G1 cells had comparable levels of pRB-associated myoblasts (Huh et al., 2004) does induce apoptosis, but this is E2F1 (Figure 1C), but the fraction of G0/G1 cells in the enriched specifically observed in the differentiating cells. Thus, taken populations differed by 14-fold (5% versus 70%). Thus, these together, these mouse studies support two general conclusions: data show that pRB-E2F1 complexes can form in S/G2/M phase first, in many different settings, Rb inactivation can induce inap- cells in response to DNA damage. propriate proliferation without triggering apoptosis, and second, pRB is sequentially phosphorylated by the cyclin D-Cdk4/6 when apoptosis is observed, it seems to result from an inability to and cyclin E-Cdk2 complexes, and this is thought to disrupt cease proliferation and undergo terminal differentiation. the interaction between pRB and E2F. Since DNA damage pRB’s apoptotic role has also been analyzed in established causes pRB to bind to E2F1 irrespective of cell-cycle phase, tissue culture cell lines. However, these studies have yielded we wished to determine whether ppRB could participate in this conflicting results: some conclude that pRB suppresses complex. To this end, we generated enriched populations of apoptosis (Almasan et al., 1995; Bosco et al., 2004; Knudsen G0/G1 and proliferating T98G cells, exposed them to either et al., 2000), whereas others suggest that it is proapoptotic (Araki ionizing radiation (IR) or doxorubicin (see schema in Figure 1D, et al., 2008; Bowen et al., 1998, 2002; Knudsen et al., 1999). left panel), and then assessed both the levels and E2F1-binding None of these studies addresses the molecular basis for the properties of ppRB using antibodies that specifically
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